Radiation induced cell death, inflammation cell death and multiple organ dysfunction necessitates employment of multiple sequential therapeutic interventions for effective radiomitigation. Our previously established oxidation of a mitochondria-specific phospholipid, cardiolipin (CL), as a required step in apoptosis resulted in the design of three new classes of mitochondria-targeted early stage radiomitigators. Recently, we discovered a new mitochondrial regulatory pathway for the CL-dependent production of lipid mediators activated in vivo during acute radiation injury. In this project, our central hypothesis is: Radiation induced systemic responses involve a series of Ca2+-independent consecutive reactions of CL oxidation and hydrolysis leading to the formation of regulatory lipid mediators with critical roles in radiation injury. These reactions include enzymatic oxidation of polyunsaturated CL species. The latter undergo hydrolysis by Ca -independent phospholipases A2 (PLA2) - 2+ iPLA2-? or LpPLA2 - to produce diversified FFAox as well as oxygenated and non-oxygenated lyso-CLs ? lipid mediators of systemic response. Three Specific Aims have been developed: In Specific Aim 1 we will use oxidative lipidomics to identify and quantitatively characterize molecular diversification of CL oxidation products and CLox-derived lipid mediators ? FFAox and lyso-CL ? in tissues and plasma at critical stages of radiation injury - apoptotic/necrotic cell death, breakage of GI epithelial barrier and penetration of bacterial pathogens into circulation and inflammatory response - culminating in multiple organ dysfunction in w/t and transgenic mice (iPLA2-? k/o, LpPLA2 k/o, Gpx4 conditional k/o) exposed to three doses of TBI. Specific Aim 2 will determine the effectiveness of small molecule inhibitors of enzymatic CLox hydrolysis by iPLA2-? and LpPLA2 in: i) suppressing the production of lipid mediators, ii) affecting critical stages of systemic response to irradiation, iii) mitigating radiation injury. Specific Aim 3 will establish the molecular mechanisms through which a specific lipid mediator of neutrophils with anti-bacterial activity, Hepoxilin A3 (HXA3), ? affects radiation damage of GI epithelium and determine the optimal timing for control of HXA3 production using small molecule regulators with maximal radiomitigative effect. These highly innovative approaches combining analytical power of oxidative lipidomics with characterization of diversified systemic responses as well as genetic manipulations and pharmacological interventions will lead to the discovery of new classes of delayed radiomiomitigators.